Overexpression of uPA-uPAR pathway components is strongly prognostic of disease recurrence and overall survival of patients with many different kinds of cancer. Because of its direct involvement in tumor malignancy, considerable interest has developed in understanding the mechanisms by which breast tumor cells exploit the uPA system to promote tumor recurrence and metastasis, and in the development of therapeutic agents that can interfere with uPA activity. Our recent and unexpected observations suggest that uPA becomes enzymatically activated intracellularly. A series of hwo dozen amiloride-based compounds have been developed capable of inhibiting intracellular or extracellular uPA activity in breast and brain tumor cells. Surprisingly, forms of the drugs that are excluded from the cell interior and act only on surface uPA are cytostatic, while their congeners capable of crossing the plasma membrane and acting on intracellular uPA induce the apoptosis of invasive tumor cells but do not affect normal tissue. These observations point to the novel notion that malignant tumor cells can become reliant on intracellular uPA for their survival. Moreover, since intracellular uPA activation is not observed within cells in normal tissue, these observations suggest that intracellular uPA is an Achilles'heel of malignant breast tumor cells that can be targeted for therapeutic intervention. Therefore, it is proposed that cell permeant, amiloride-based uPA inhibitors cause apoptosis of proliferative and infiltrative breast cancer cells by a novel intracellular mechanism that disrupts autocrine regulation ofthe uPA signaling system. Furthermore, the administration of these compounds to a transgenic mouse model of recurrent breast cancer should result in a suppression of tumor metastasis and recurrence. Aim 1: Analyze molecular mechanisms underlying drug-induced apoptosis in cultured breast cancer cells. Aim 2: Determine the impact of uPA inhibitors on tumor development, growth, metastasis and recurrence in atransgenic mouse model of breast cancer. Public Health Relevance: The major challenge in developing novel and effective treatments for breast cancer is the identification of druggable targets that play major roles in the malignancy of breast tumors, but play minor roles in other physiological processes. Therapeutic inhibition of such targets will specifically thwart tumor cells while eliciting minimal patient side effects. Since intracellular uPA activation is unique to malignant tumor cells, and because tumor cell survival appears to be dependent on this mechanism, inhibitors of intracellular uPA offer tremendous potential in the treatment of the most aggressive forms of breast cancer.